G protein-coupled receptors (GPCR) play a crucial role in the regulation of the immune response by, e.g., chemokines, PGs, and β2-adrenergic agonists. The responsiveness of these GPCRs is turned off by the family of G protein-coupled receptor kinases (GRK1–6). These kinases act by phosphorylating the GPCR in an agonist-dependent manner, resulting in homologous desensitization of the receptor. Although GRKs are widely expressed throughout the body, leukocytes express relatively high levels of GRKs, in particular GRK2, -3, and -6. We investigated whether in vivo the inflammatory disease adjuvant arthritis (AA) induces changes in GRK expression and function in the immune system. In addition, we analyzed whether the systemic effects of AA also involve changes in GRKs in nonimmune organs. At the peak of the inflammatory process, we observed a profound down-regulation of GRK2, -3, and -6 in splenocytes and mesenteric lymph node cells from AA rats. Interestingly, no changes in GRK were observed in thymocytes and in nonimmune organs such as heart and pituitary. During the remission phase of AA, GRK levels in spleen and mesenteric lymph nodes are returning to baseline levels. The decrease in GRK2 at the peak of AA is restricted to CD45RA+ B cells and CD4+ T cells, and was not observed in CD8+ T cells. In conclusion, we demonstrate in this study, for the first time, that an inflammatory process in vivo induces a tissue-specific down-regulation of GRKs in the immune system.

Chemokine receptors belong to the family of G-protein-coupled receptors (GPCR). Phosphorylation of GPCR by GPCR kinases (GRKs) is considered to play an important role in desensitization of these receptors. We have recently shown in patients with rheumatoid arthritis that the level of GRK2 in lymphocytes is reduced by approximately 50%. However, the physiological relevance of reduced GRK2 levels in lymphocytes is not known. Here, we investigated whether reduced GRK2 expression changes the chemotactic response of T cells to the chemokines CCL3, CCL4, and CCL5. Activated T cells from GRK2+/- mice, which have a 50% reduction in GRK2 protein levels, showed a significant 40% increase in chemotaxis toward the CCR5 ligand CCL4. In addition, chemotaxis toward the CCR1 and CCR5 ligands CCL3 and CCL5 was also increased. Binding of CCL4 to activated T cells from GRK2+/- and wild-type (WT) mice was similar, but agonist-induced CCR5 phosphorylation was attenuated in GRK2+/- cells. Moreover, the calcium response and phosphorylation of protein kinase B and extracellular-regulated kinase in response to CCL4 were significantly increased in GRK2+/- T cells, showing that signaling is increased when the level of GRK2 is reduced. GRK2+/- and WT cells do become refractory to restimulation with CCL4. In conclusion, a 50% decrease in T cell GRK2 expression results in increased responsiveness to CCL3, CCL4, and CCL5, suggesting that the 50% reduction in lymphocyte GRK2 level as observed during inflammation can have functional consequences for the response of these cells to chemokines.

IntroductionResults from clinical studies have provided evidence for the importance of leukocyte-endothelial interactions in the pathogenesis of pulmonary diseases such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), as well as in systemic events like sepsis and multiple organ failure (MOF). The present study was designed to investigate whether alveolar stretch due to mechanical ventilation (MV) may evoke endothelial activation and inflammation in healthy mice, not only in the lung but also in organs distal to the lung.MethodsHealthy male C3H/HeN mice were anesthetized, tracheotomized and mechanically ventilated for either 1, 2 or 4 hours. To study the effects of alveolar stretch in vivo, we applied a MV strategy that causes overstretch of pulmonary tissue i.e. 20 cmH2O peak inspiratory pressure (PIP) and 0 cmH20 positive end expiratory pressure (PEEP). Non-ventilated, sham-operated animals served as a reference group (non-ventilated controls, NVC).ResultsAlveolar stretch imposed by MV did not only induce de novo synthesis of adhesion molecules in the lung but also in organs distal to the lung, like liver and kidney. No activation was observed in the brain. In addition, we demonstrated elevated cytokine and chemokine expression in pulmonary, hepatic and renal tissue after MV which was accompanied by enhanced recruitment of granulocytes to these organs.ConclusionsOur data implicate that MV causes endothelial activation and inflammation in mice without pre-existing pulmonary injury, both in the lung and distal organs.

Major concern has emerged about the possible long term adverse effects of glucocorticoid treatment, which is frequently used for the prevention of chronic lung disease in preterm infants. Here we show that neonatal glucocorticoid treatment of rats increases the severity (p ≤ 0.01) and incidence (p ≤ 0.01) of the inflammatory autoimmune disease experimental autoimmune encephalomyelitis in adult life. In search of possible mechanisms responsible for the increased susceptibility to experimental autoimmune encephalomyelitis, we investigated the reactivity of the hypothalamo-pituitary-adrenal axis and of immune cells in adult rats after neonatal glucocorticoid treatment. We observed that neonatal glucocorticoid treatment reduces the corticosterone response after an LPS challenge in adult rats (p ≤ 0.001). Interestingly, LPS-stimulated macrophages of glucocorticoid-treated rats produce less TNF-α and IL-1β in adult life than control rats (p < 0.05). In addition, splenocytes obtained from adult rats express increased mRNA levels of the proinflammatory cytokines IFN-γ (p < 0.01) and TNF-β (p < 0.05) after neonatal glucocorticoid treatment. Apparently, neonatal glucocorticoid treatment has permanent programming effects on endocrine as well as immune functioning in adult life. In view of the frequent clinical application of glucocorticoids to preterm infants, our data demonstrate that neonatal glucocorticoid treatment may be a risk factor for the development of (auto)immune disease in man.

BackgroundLoss of integrity of the epithelial and endothelial barriers is thought to be a prominent feature of ventilator-induced lung injury (VILI). Based on its function in vascular integrity, we hypothesize that the angiopoietin (Ang)-Tie2 system plays a role in the development of VILI. The present study was designed to examine the effects of mechanical ventilation on the Ang-Tie2 system in lung tissue. Moreover, we evaluated whether treatment with Ang-1, a Tie2 receptor agonist, protects against inflammation, vascular leakage and impaired gas exchange induced by mechanical ventilation.MethodsMice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with either an inspiratory pressure of 10 cmH2O (‘low’ tidal volume ∼7.5 ml/kg; LVT) or 18 cmH2O (‘high’ tidal volume ∼15 ml/kg; HVT). At initiation of HVT-ventilation, recombinant human Ang-1 was intravenously administered (1 or 4 µg per animal). Non-ventilated mice served as controls.ResultsHVT-ventilation influenced the Ang-Tie2 system in lungs of healthy mice since Ang-1, Ang-2 and Tie2 mRNA were decreased. Treatment with Ang-1 increased Akt-phosphorylation indicating Tie2 signaling. Ang-1 treatment reduced infiltration of granulocytes and expression of keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1 and interleukin (IL)-1β caused by HVT-ventilation. Importantly, Ang-1 treatment did not prevent vascular leakage and impaired gas exchange in HVT-ventilated mice despite inhibition of inflammation, vascular endothelial growth factor (VEGF) and Ang-2 expression.ConclusionsAng-1 treatment downregulates pulmonary inflammation, VEGF and Ang-2 expression but does not protect against vascular leakage and impaired gas exchange induced by HVT-ventilation.

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BACKGROUND AND PURPOSESystemic glucocorticoid therapy may effectively attenuate lung inflammation but also induce severe side-effects. Delivery of glucocorticoids by liposomes could therefore be beneficial. We investigated if liposome-encapsulated dexamethasone inhibited ventilator-induced lung inflammation. Furthermore, we evaluated whether targeting of cellular Fcg-receptors (FcgRs) by conjugating immunoglobulin G (IgG) to liposomes, would improve the efficacy of dexamethasone-liposomes in attenuating granulocyte infiltration, one of the hallmarks of lung inflammation. EXPERIMENTAL APPROACHMice were anaesthetized, tracheotomized and mechanically ventilated for 5 h with either 'low' tidal volumes~7.5 mL·kg -1 (LVT) or 'high' tidal volumes~15 mL·kg -1 (HVT). At initiation of ventilation, we intravenously administered dexamethasone encapsulated in liposomes (Dex-liposomes), dexamethasone encapsulated in IgG-modified liposomes (IgG-Dex-liposomes) or free dexamethasone. Non-ventilated mice served as controls. KEY RESULTSDex-liposomes attenuated granulocyte infiltration and IL-6 mRNA expression after LVT-ventilation, but not after HVT-ventilation. Dex-liposomes also down-regulated mRNA expression of IL-1b and KC, but not of CCL2 (MCP-1) in lungs of LVT and HVT-ventilated mice. Importantly, IgG-Dex-liposomes inhibited granulocyte influx caused by either LVT or HVTventilation. IgG-Dex-liposomes diminished IL-1b and KC mRNA expression in both ventilation groups, and IL-6 and CCL2 mRNA expression in the LVT-ventilated group. Free dexamethasone prevented granulocyte influx and inflammatory mediator expression induced by LVT or HVT-ventilation. CONCLUSIONS AND IMPLICATIONSFcgR-targeted IgG-Dex-liposomes are pharmacologically more effective than Dex-liposomes particularly in inhibiting pulmonary granulocyte infiltration. IgG-Dex-liposomes inhibited most parameters of ventilator-induced lung inflammation as effectively as free dexamethasone, with the advantage that liposome-encapsulated dexamethasone will be released locally in the lung thereby preventing systemic side-effects. AbbreviationsALI, acute lung

IntroductionAneurysmal subarachnoid hemorrhage (SAH) affects relatively young people and carries a poor prognosis with a case fatality rate of 35%. One of the major systemic complications associated with SAH is acute lung injury (ALI) which occurs in up to one-third of the patients and is associated with poor outcome. ALI in SAH may be predisposed by neurogenic pulmonary edema (NPE) and inflammatory mediators. The objective of this study was to assess the immunomodulatory effects of interferon-β (IFN-β) on inflammatory mediators in the lung after experimental SAH.MethodsMale Wistar rats were subjected to the induction of SAH by means of the endovascular filament method. Sham-animals underwent sham-surgery. Rats received IFN-β for four consecutive days starting at two hours after SAH induction. After seven days, lungs were analyzed for the expression of inflammatory markers.ResultsSAH induced the influx of neutrophils into the lung, and enhanced expression of the pulmonary adhesion molecules E-selectin, inter-cellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 compared to sham-animals. In addition, SAH increased the expression of the chemokines macrophage inflammatory protein (MIP)-1α, MIP-2, and cytokine-induced neutrophil chemoattractant (CINC)-1 in the lung. Finally, tumor necrosis factor-α (TNF-α) was significantly increased in lungs from SAH-animals compared to sham-animals. IFN-β effectively abolished the SAH-induced expression of all pro-inflammatory mediators in the lung.ConclusionsIFN-β strongly reduces lung inflammation after experimental SAH and may therefore be an effective drug to prevent SAH-mediated lung injury.